Although membrane-containing dsDNA bacterial viruses are some of the most prevalent predators in aquatic environments, we know little about how they function due to their intractability in the laboratory. Here, we have identified and thoroughly characterized a new type of membrane-containing bacteriophage, Jorvik, that infects the freshwater mixotrophic model bacterium Rhodobacter capsulatus. Jorvik is extremely virulent, can persist in the host integrated into the RuBisCo operon and encodes two experimentally verified cell wall hydrolases. Jorvik-like prophages are abundant in the genomes of Alphaproteobacteria, are distantly related to known viruses of the class Tectiliviricetes, and we propose they should be classified as a new family. Crucially, we demonstrate how widely used phage manipulation methods should be adjusted to prevent loss of virus infectivity. Our thorough characterization of environmental phage Jorvik provides important experimental insights about phage diversity and interactions in microbial communities that are often unexplored in common metagenomic analyses.

Biology Department University of York Wentworth Way York YO10 5DD UK

Department of Experimental Biology Faculty of Science Masaryk University 625 00 Brno Czech Republic

York Biomedical Research Institute University of York Wentworth Way York YO10 5NG UK

York Structural Biology Laboratory Department of Chemistry University of York Heslington York YO10 5DD UK

Zobrazit více v PubMed

Mäntynen S., Sundberg L.-R., Oksanen H.M., Poranen M.M. Half a Century of Research on Membrane-Containing Bacteriophages: Bringing New Concepts to Modern Virology. Viruses. 2019;11:76. doi: 10.3390/v11010076. PubMed DOI PMC

Krishnamurthy S.R., Janowski A.B., Zhao G., Barouch D., Wang D. Hyperexpansion of RNA Bacteriophage Diversity. PLoS Biol. 2016;14:e1002409. doi: 10.1371/journal.pbio.1002409. PubMed DOI PMC

Hopkins M., Kailasan S., Cohen A., Roux S., Tucker K.P., Shevenell A., Agbandje-McKenna M., Breitbart M. Diversity of environmental single-stranded DNA phages revealed by PCR amplification of the partial major capsid protein. ISME J. 2014;8:2093–2103. doi: 10.1038/ismej.2014.43. PubMed DOI PMC

Kivelä H.M., Männistö R.H., Kalkkinen N., Bamford D.H. Purification and Protein Composition of PM2, the First Lipid-Containing Bacterial Virus To Be Isolated. Virology. 1999;262:364–374. doi: 10.1006/viro.1999.9838. PubMed DOI

Kauffman K.M., Hussain F.A., Yang J., Arevalo P., Brown J.M., Chang W.K., VanInsberghe D., Elsherbini J., Sharma R.S., Cutler M.B., et al. A major lineage of non-tailed dsDNA viruses as unrecognized killers of marine bacteria. Nature. 2018;554:118–122. doi: 10.1038/nature25474. PubMed DOI

Espejo R.T., Canelo E.S. Properties of bacteriophage PM2: a lipid-containing bacterial virus. Virology. 1968;34:738–747. doi: 10.1016/0042-6822(68)90094-9. PubMed DOI

Kivelä H.M., Kalkkinen N., Bamford D.H. Bacteriophage PM2 Has a Protein Capsid Surrounding a Spherical Proteinaceous Lipid Core. J. Virol. 2002;76:8169–8178. doi: 10.1128/JVI.76.16.8169-8178.2002. PubMed DOI PMC

Brum J.R., Schenck R.O., Sullivan M.B. Global morphological analysis of marine viruses shows minimal regional variation and dominance of non-tailed viruses. ISME J. 2013;7:1738–1751. doi: 10.1038/ismej.2013.67. PubMed DOI PMC

Yutin N., Bäckström D., Ettema T.J.G., Krupovic M., Koonin E.V. Vast diversity of prokaryotic virus genomes encoding double jelly-roll major capsid proteins uncovered by genomic and metagenomic sequence analysis. Virol. J. 2018;15:67. doi: 10.1186/s12985-018-0974-y. PubMed DOI PMC

Abrescia N.G.A., Bamford D.H., Grimes J.M., Stuart D.I. Structure unifies the viral universe. Annu. Rev. Biochem. 2012;81:795–822. doi: 10.1146/annurev-biochem-060910-095130. PubMed DOI

Woo A.C., Gaia M., Guglielmini J., Da Cunha V., Forterre P. Phylogeny of the Varidnaviria Morphogenesis Module: Congruence and Incongruence With the Tree of Life and Viral Taxonomy. Front. Microbiol. 2021;12:704052. doi: 10.3389/fmicb.2021.704052. PubMed DOI PMC

Bamford D.H. Do viruses form lineages across different domains of life? Res. Microbiol. 2003;154:231–236. doi: 10.1016/S0923-2508(03)00065-2. PubMed DOI

Azinas S., Bano F., Torca I., Bamford D.H., Schwartz G.A., Esnaola J., Oksanen H.M., Richter R.P., Abrescia N.G. Membrane-containing virus particles exhibit the mechanics of a composite material for genome protection. Nanoscale. 2018;10:7769–7779. doi: 10.1039/C8NR00196K. PubMed DOI PMC

Walker P.J., Siddell S.G., Lefkowitz E.J., Mushegian A.R., Adriaenssens E.M., Dempsey D.M., Dutilh B.E., Harrach B., Harrison R.L., Hendrickson R.C., et al. Changes to virus taxonomy and the Statutes ratified by the International Committee on Taxonomy of Viruses (2020) Arch. Virol. 2020;165:2737–2748. doi: 10.1007/s00705-020-04752-x. PubMed DOI

Peralta B., Gil-Carton D., Castaño-Díez D., Bertin A., Boulogne C., Oksanen H.M., Bamford D.H., Abrescia N.G.A. Mechanism of Membranous Tunnelling Nanotube Formation in Viral Genome Delivery. PLoS Biol. 2013;11:e1001667. doi: 10.1371/journal.pbio.1001667. PubMed DOI PMC

Krupovič M., Bamford D.H. Putative prophages related to lytic tailless marine dsDNA phage PM2 are widespread in the genomes of aquatic bacteria. BMC Genom. 2007;8:236. doi: 10.1186/1471-2164-8-236. PubMed DOI PMC

Joo Y.J., Kim H.-J., Lee J.Y., Kim J. Biochemical quantitation of PM2 phage DNA as a substrate for endonuclease assay. J. Microbiol. 2004;42:99–102. PubMed

Abrescia N.G.A., Grimes J.M., Kivelä H.M., Assenberg R., Sutton G.C., Butcher S.J., Bamford J.K.H., Bamford D.H., Stuart D.I. Insights into Virus Evolution and Membrane Biogenesis from the Structure of the Marine Lipid-Containing Bacteriophage PM2. Mol. Cell. 2008;31:749–761. doi: 10.1016/j.molcel.2008.06.026. PubMed DOI

Laanto E., Mäntynen S., De Colibus L., Marjakangas J., Gillum A., Stuart D.I., Ravantti J.J., Huiskonen J.T., Sundberg L.-R. Virus found in a boreal lake links ssDNA and dsDNA viruses. Proc. Natl. Acad. Sci. 2017;114:8378–8383. doi: 10.1073/pnas.1703834114. PubMed DOI PMC

Kejzar N., Laanto E., Rissanen I., Abrishami V., Selvaraj M., Moineau S., Ravantti J., Sundberg L.-R., Huiskonen J.T. Cryo-EM structure of ssDNA bacteriophage ΦCjT23 provides insight into early virus evolution. Nat. Commun. 2022;13:7478. doi: 10.1038/s41467-022-35123-6. PubMed DOI PMC

Kalatzis P.G., Mauritzen J.J., Winther-Have C.S., Michniewski S., Millard A., Tsertou M.I., Katharios P., Middelboe M. Staying below the Radar: Unraveling a New Family of Ubiquitous “Cryptic” Non-Tailed Temperate Vibriophages and Implications for Their Bacterial Hosts. Int. J. Mol. Sci. 2023;24:3937. doi: 10.3390/ijms24043937. PubMed DOI PMC

Strnad H., Lapidus A., Paces J., Ulbrich P., Vlcek C., Paces V., Haselkorn R. Complete genome sequence of the photosynthetic purple nonsulfur bacterium Rhodobacter capsulatus SB 1003. J. Bacteriol. 2010;192:3545–3546. doi: 10.1128/JB.00366-10. PubMed DOI PMC

Ding H., Moksa M.M., Hirst M., Beatty J.T. Draft Genome Sequences of Six Rhodobacter capsulatus Strains, YW1, YW2, B6, Y262, R121, and DE442. Genome Announc. 2014;2:e00050-14. doi: 10.1128/genomeA.00050-14. PubMed DOI PMC

Weaver P.F., Wall J.D., Gest H. Characterization of Rhodopseudomonas capsulata. Arch. Microbiol. 1975;105:207–216. doi: 10.1007/BF00447139. PubMed DOI

Leidenfrost R.M., Wappler N., Wünschiers R. Draft Genome Assembly of Rhodobacter sphaeroides 2.4.1 Substrain H2 from Nanopore Data. Microbiol. Resour. Announc. 2020;9:e00414-20. doi: 10.1128/MRA.00414-20. PubMed DOI PMC

Wall J.D., Weaver P.F., Gest H. Gene transfer agents, bacteriophages, and bacteriocins of Rhodopseudomonas capsulata. Arch. Microbiol. 1975;105:217–224. doi: 10.1007/BF00447140. PubMed DOI

Duyvesteyn H.M.E., Ginn H.M., Pietilä M.K., Wagner A., Hattne J., Grimes J.M., Hirvonen E., Evans G., Parsy M.-L., Sauter N.K., et al. Towards in cellulo virus crystallography. Sci. Rep. 2018;8:3771. doi: 10.1038/s41598-018-21693-3. PubMed DOI PMC

Kivelä H.M., Daugelavičius R., Hankkio R.H., Bamford J.K.H., Bamford D.H. Penetration of Membrane-Containing Double-Stranded-DNA Bacteriophage PM2 into Pseudoalteromonas Hosts. J. Bacteriol. 2004;186:5342–5354. doi: 10.1128/JB.186.16.5342-5354.2004. PubMed DOI PMC

Gudlavalleti B.S., Phung T., Barton C.L., Becker A., Graul B.L., Griffin J.T., Hays C.J., Horn B., Liang D.R., Rutledge L.M., et al. Whole genome sequencing identifies an allele responsible for clear vs. turbid plaque morphology in a Mycobacteriophage. BMC Microbiol. 2020;20:148. doi: 10.1186/s12866-020-01833-4. PubMed DOI PMC

Bondy-Denomy J., Qian J., Westra E.R., Buckling A., Guttman D.S., Davidson A.R., Maxwell K.L. Prophages mediate defense against phage infection through diverse mechanisms. ISME J. 2016;10:2854–2866. doi: 10.1038/ismej.2016.79. PubMed DOI PMC

Carnoy C., Roten C.-A. The dif/Xer Recombination Systems in Proteobacteria. PLoS One. 2009;4:e6531. doi: 10.1371/journal.pone.0006531. PubMed DOI PMC

Huber K.E., Waldor M.K. Filamentous phage integration requires the host recombinases XerC and XerD. Nature. 2002;417:656–659. doi: 10.1038/nature00782. PubMed DOI

Brimacombe C.A., Ding H., Beatty J.T. Rhodobacter capsulatus DprA is essential for RecA-mediated gene transfer agent (RcGTA) recipient capability regulated by quorum-sensing and the CtrA response regulator. Mol. Microbiol. 2014;92:1260–1278. doi: 10.1111/mmi.12628. PubMed DOI

Fogg P.C.M. Identification and characterization of a direct activator of a gene transfer agent. Nat. Commun. 2019;10:595. doi: 10.1038/s41467-019-08526-1. PubMed DOI PMC

Mascolo E., Adhikari S., Caruso S.M., deCarvalho T., Folch Salvador A., Serra-Sagristà J., Young R., Erill I., Curtis P.D. The transcriptional regulator CtrA controls gene expression in Alphaproteobacteria phages: Evidence for a lytic deferment pathway. Front. Microbiol. 2022;13:918015. doi: 10.3389/fmicb.2022.918015. PubMed DOI PMC

Kropinski A.M. Measurement of the rate of attachment of bacteriophage to cells. Methods Mol. Biol. 2009;501:151–155. doi: 10.1007/978-1-60327-164-6_15. PubMed DOI

Storms Z.J., Arsenault E., Sauvageau D., Cooper D.G. Bacteriophage adsorption efficiency and its effect on amplification. Bioprocess Biosyst. Eng. 2010;33:823–831. doi: 10.1007/s00449-009-0405-y. PubMed DOI

Bamford D.H., Rouhiainen L., Takkinen K., Söderlund H. Comparison of the Lipid-containing Bacteriophages PRD1, PR3, PR4, PR5 and L17. J. Gen. Virol. 1981;57:365–373. doi: 10.1099/0022-1317-57-2-365. PubMed DOI

Zhang Y., Jiao N. Roseophage RDJLΦ1, Infecting the Aerobic Anoxygenic Phototrophic Bacterium Roseobacter denitrificans OCh114. Appl. Environ. Microbiol. 2009;75:1745–1749. doi: 10.1128/AEM.02131-08. PubMed DOI PMC

Meier-Kolthoff J.P., Göker M. VICTOR: genome-based phylogeny and classification of prokaryotic viruses. Bioinformatics. 2017;33:3396–3404. doi: 10.1093/bioinformatics/btx440. PubMed DOI PMC

Zimmermann L., Stephens A., Nam S.-Z., Rau D., Kübler J., Lozajic M., Gabler F., Söding J., Lupas A.N., Alva V. A Completely Reimplemented MPI Bioinformatics Toolkit with a New HHpred Server at its Core. J. Mol. Biol. 2018;430:2237–2243. doi: 10.1016/j.jmb.2017.12.007. PubMed DOI

Yutin N., Rayko M., Antipov D., Mutz P., Wolf Y.I., Krupovic M., Koonin E.V. Varidnaviruses in the Human Gut: A Major Expansion of the Order Vinavirales. Viruses. 2022;14:1842. doi: 10.3390/v14091842. PubMed DOI PMC

Hyman P. Phages for Phage Therapy: Isolation, Characterization, and Host Range Breadth. Pharmaceuticals. 2019;12:35. doi: 10.3390/ph12010035. PubMed DOI PMC

Poxleitner M., Pope W., Jacobs-Sera D., Sivanathan V., Hatfull G. Chapter 5: Isolation. Sea Phage Discovery Guide. Howard Hughes Medical Institute; 2018. https://seaphagesphagediscoveryguide.helpdocsonline.com/5-0-toc

Sharma S., Datta S., Chatterjee S., Dutta M., Samanta J., Vairale M.G., Gupta R., Veer V., Dwivedi S.K. Isolation and characterization of a lytic bacteriophage against Pseudomonas aeruginosa. Sci. Rep. 2021;11:19393. doi: 10.1038/s41598-021-98457-z. PubMed DOI PMC

Henry M., Lavigne R., Debarbieux L. Predicting In Vivo Efficacy of Therapeutic Bacteriophages Used To Treat Pulmonary Infections. Antimicrob. Agents Chemother. 2013;57:5961–5968. doi: 10.1128/AAC.01596-13. PubMed DOI PMC

Brimacombe C.A., Stevens A., Jun D., Mercer R., Lang A.S., Beatty J.T. Quorum-sensing regulation of a capsular polysaccharide receptor for the Rhodobacter capsulatus gene transfer agent (RcGTA) Mol. Microbiol. 2013;87:802–817. doi: 10.1111/mmi.12132. PubMed DOI PMC

Sherlock D., Fogg P.C.M. Loss of the Rhodobacter capsulatus Serine Acetyl Transferase Gene, cysE1, Impairs Gene Transfer by Gene Transfer Agents and Biofilm Phenotypes. Appl. Environ. Microbiol. 2022;88:e0094422. doi: 10.1128/aem.00944-22. PubMed DOI PMC

Bazinet C.W., King J. A late gene product of phage P22 affecting virus infectivity. Virology. 1985;143:368–379. doi: 10.1016/0042-6822(85)90377-0. PubMed DOI

Westbye A.B., Leung M.M., Florizone S.M., Taylor T.A., Johnson J.A., Fogg P.C., Beatty J.T. Phosphate concentration and the putative sensor kinase protein CckA modulate cell lysis and release of the Rhodobacter capsulatus gene transfer agent. J. Bacteriol. 2013;195:5025–5040. doi: 10.1128/JB.00669-13. PubMed DOI PMC

Männistö R.H., Grahn A.M., Bamford D.H., Bamford J.K.H. Transcription of Bacteriophage PM2 Involves Phage-Encoded Regulators of Heterologous Origin. J. Bacteriol. 2003;185:3278–3287. doi: 10.1128/JB.185.11.3278-3287.2003. PubMed DOI PMC

Benešík M., Nováček J., Janda L., Dopitová R., Pernisová M., Melková K., Tišáková L., Doškař J., Žídek L., Hejátko J., Pantůček R. Role of SH3b binding domain in a natural deletion mutant of Kayvirus endolysin LysF1 with a broad range of lytic activity. Virus Genes. 2018;54:130–139. doi: 10.1007/s11262-017-1507-2. PubMed DOI

Rodríguez-Rubio L., Martínez B., Donovan D.M., Rodríguez A., García P. Bacteriophage virion-associated peptidoglycan hydrolases: potential new enzybiotics. Crit. Rev. Microbiol. 2013;39:427–434. doi: 10.3109/1040841X.2012.723675. PubMed DOI

Rydman P.S., Bamford D.H. The Lytic Enzyme of Bacteriophage PRD1 Is Associated with the Viral Membrane. J. Bacteriol. 2002;184:104–110. doi: 10.1128/JB.184.1.104-110.2002. PubMed DOI PMC

Fogg M.J., Wilkinson A.J. Higher-throughput approaches to crystallization and crystal structure determination. Biochem. Soc. Trans. 2008;36:771–775. doi: 10.1042/BST0360771. PubMed DOI

Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014;30:2068–2069. doi: 10.1093/bioinformatics/btu153. PubMed DOI

Besemer J., Lomsadze A., Borodovsky M. GeneMarkS: a self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions. Nucleic Acids Res. 2001;29:2607–2618. doi: 10.1093/nar/29.12.2607. PubMed DOI PMC

Carver T., Harris S.R., Berriman M., Parkhill J., McQuillan J.A. Artemis: an integrated platform for visualization and analysis of high-throughput sequence-based experimental data. Bioinformatics. 2012;28:464–469. doi: 10.1093/bioinformatics/btr703. PubMed DOI PMC

Altschul S.F., Madden T.L., Schäffer A.A., Zhang J., Zhang Z., Miller W., Lipman D.J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25:3389–3402. doi: 10.1093/nar/25.17.3389. PubMed DOI PMC

Jumper J., Evans R., Pritzel A., Green T., Figurnov M., Ronneberger O., Tunyasuvunakool K., Bates R., Žídek A., Potapenko A., et al. Highly accurate protein structure prediction with AlphaFold. Nature. 2021;596:583–589. doi: 10.1038/s41586-021-03819-2. PubMed DOI PMC

Mirdita M., Schütze K., Moriwaki Y., Heo L., Ovchinnikov S., Steinegger M. ColabFold: making protein folding accessible to all. Nat. Methods. 2022;19:679–682. doi: 10.1038/s41592-022-01488-1. PubMed DOI PMC

Holm L. DALI and the persistence of protein shape. Protein Sci. 2020;29:128–140. doi: 10.1002/pro.3749. PubMed DOI PMC

Kans J. Entrez Direct: E-utilities on the Unix Command Line. Entrez Programming Utilities Help. NCBI; 2022. pp. 85–186.https://www.ncbi.nlm.nih.gov/books/NBK179288

Katoh K., Standley D.M. MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Mol. Biol. Evol. 2013;30:772–780. doi: 10.1093/molbev/mst010. PubMed DOI PMC

Capella-Gutiérrez S., Silla-Martínez J.M., Gabaldón T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics. 2009;25:1972–1973. doi: 10.1093/bioinformatics/btp348. PubMed DOI PMC

Minh B.Q., Nguyen M.A.T., von Haeseler A. Ultrafast Approximation for Phylogenetic Bootstrap. Mol. Biol. Evol. 2013;30:1188–1195. doi: 10.1093/molbev/mst024. PubMed DOI PMC

Kremer J.R., Mastronarde D.N., McIntosh J.R. Computer Visualization of Three-Dimensional Image Data Using IMOD. J. Struct. Biol. 1996;116:71–76. doi: 10.1006/jsbi.1996.0013. PubMed DOI

Goddard T.D., Huang C.C., Meng E.C., Pettersen E.F., Couch G.S., Morris J.H., Ferrin T.E. UCSF ChimeraX: Meeting modern challenges in visualization and analysis. Protein Sci. 2018;27:14–25. doi: 10.1002/pro.3235. PubMed DOI PMC

Wickham H. Springer; 2016. ggplot2, Elegant Graphics for Data Analysis.

Lee I., Ouk Kim Y., Park S.C., Chun J. OrthoANI: an improved algorithm and software for calculating average nucleotide identity. Int. J. Syst. Evol. Microbiol. 2016;66:1100–1103. doi: 10.1099/ijsem.0.000760. PubMed DOI

Ormerod J.G., Ormerod K.S., Gest H. Light-dependent utilization of organic compounds and photoproduction of molecular hydrogen by photosynthetic bacteria; relationships with nitrogen metabolism. Arch. Biochem. Biophys. 1961;94:449–463. doi: 10.1016/0003-9861(61)90073-x. PubMed DOI

Kropinski A.M. Practical Advice on the One-Step Growth Curve. Methods Mol. Biol. 2018;1681:41–47. doi: 10.1007/978-1-4939-7343-9_3. PubMed DOI

Sambrook J., Russell D.W. In: Molecular cloning: A Laboratory Manual. Irwin N., Janssen K.A., editors. Vol. 8. Cold Spring Harbor Laboratory Press; 2001. Commonly used techniques in molecular cloning.

Hynes A., Lang A. Rhodobacter capsulatus Gene Transfer Agent (RcGTA) Activity Bioassays. Bio. Protoc. 2013;3:e317. doi: 10.21769/BioProtoc.317. DOI

Grybchuk D., Macedo D.H., Kleschenko Y., Kraeva N., Lukashev A.N., Bates P.A., Kulich P., Leštinová T., Volf P., Kostygov A.Y., Yurchenko V. The First Non-LRV RNA Virus in Leishmania. Viruses. 2020;12:168. doi: 10.3390/v12020168. PubMed DOI PMC

Fogg P.C.M., Westbye A.B., Beatty J.T. One for All or All for One: Heterogeneous Expression and Host Cell Lysis Are Key to Gene Transfer Agent Activity in Rhodobacter capsulatus. PLoS One. 2012;7:e43772. doi: 10.1371/journal.pone.0043772. PubMed DOI PMC

A stargate mechanism of Microviridae genome delivery unveiled by cryogenic electron tomography